Centrifugation, the use of centrifugal force to separate particles, has been used for decades. Samples comprising the particles suspended in a medium are spun in a rotor at a high rate of speed and centrifugal force causes the particles to move outwardly from the rotational center of the rotor towards the periphery. This movement is known as sedimentation. The sedimentation rate is dependent upon several factors such as the rotational speed, the density and viscosity of the medium, the density of the particle, the size and the shape of the particle. The particles are separated in space by the differing distances they traverse along a centrifugal force vector. The degree of separation along this force vector determines the degree of resolution with which particles may be separated.
In density gradient ultracentrifugation, the density of the suspension medium varies in a known manner from one end of the centrifuge tube to the other. When the particle under the influence of centrifugal force reaches the point of its isopycnic density, i.e., when the density of the surrounding liquid is equal to the density of the particle, the particle it will cease to migrate along the force vector.
Solute systems used to establish density gradients for ultracentrifugation include inorganic salts (cesium chloride, potassium bromide, sodium chloride), sucrose and several commercially available solutes such as Ficoll(copyright), a synthetic polysaccharide made by crosslinking sucrose; Percoll(trademark), a suspension of silica particles coated with polyvinylpyrrolidone; and Nycodenz(copyright), a derivative of the synthetic molecule metrizoic acid (metrizamide). Iodixinol, a dimer of Nycodenz(copyright), is also used widely.
These solute systems are plagued by several deficiencies. The inorganic salts must be used at high concentrations which may cause dehydration of biological particle analytes, thus changing the physical/chemical properties of the analytes. The solutes can also alter the physical/chemical properties of the particles by forming solvation spheres around the particles.
Sucrose and its polysaccharide derivatives tend to form highly viscous solutions at high solute densities. This dramatically increases the time required to reach sedimentation equilibrium.
Because the density of a solution is proportional to the concentration of solute, density gradients are typically formed using the above solutes by layering solutions of lower concentration on top of solutions of higher concentration. For example, a density gradient can be made by layering solutions of sucrose one on top of another to form a gradient from 10-40% sucrose from the top of the tube to the bottom. This process is time consuming and may suffer from poor reproducibility from sample to sample.
Various methods and devices for forming density gradients have been explored in an effort to improve the ease and reproducibility of using the above solute systems. For example, U.S. Pat. No. 5,171,539 describes an apparatus for generating a continuous solution gradient wherein solutions of differing concentrations are layered in a tube and the tube is disposed at an angle with respect to the vertical. The tube is rotated for a period of time thereby generating a continuous solution gradient.
U.S. Pat. No. 4,290,300 describes a device having a chamber for a heavy concentration of sucrose and a chamber for a light concentration of sucrose. The relative rate of release from the two chambers is controlled by the pressure in the chambers, thus allowing the formation of linear or exponential density gradients.
An alternate method would be to use a solute system where a density gradient xe2x80x9cself-formsxe2x80x9d when the system is exposed to a centrifugal field. U.S. Pat. No. 4,480,038 describes a self-forming gradient using 60% Percoll(trademark) containing 25 mM sucrose.
U.S. Pat. No. 5,985,037 describes a self-forming density gradient created by applying a centrifugal field to a solution that contains 27-33% Percoll(trademark) and 36-44% sugar.
These self-forming gradients are easier to reproduce, but they require a high concentration of solute. The gradient environment therefore differs significantly from physiological conditions. High solutes concentrations also increase the viscosity of the solution thereby increasing the length of time that is required for the particles to reach sedimentation equilibrium. Further, the availability of a self-forming density gradient that avoids the use of silica-based solutes is desirable because silica-based solutes do not mimic physiological conditions.
One aspect of the present invention is a method of forming a density gradient, the method comprising: providing a solution of one or more metal ion chelate complexes and applying a centrifugal field to the solution until a density gradient is formed.
A further aspect of the invention is a method of separating particles according to their density, the method comprising: providing a composition comprising the particles and a solution of one or more metal ion chelate complexes; and applying a centrifugal field to the composition until the solution has formed a density gradient and the particles have partitioned along the density gradient.
A still further aspect of the present invention is a density gradient comprising one or more metal ion chelate complexes.